Methods of Treating CMV Retinitis by T Cell Therapy

ABSTRACT

Disclosed herein are methods of treating CMV (cytomegalovirus) retinitis in a human patient in need thereof, comprising administering to the human patient a population of allogeneic T cells comprising CMV-specific T cells, wherein the human patient is infected with HIV or has been the recipient of a solid organ transplant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Nos. 62/185,558, filed Jun. 26, 2015, and 62/191,304, filedJul. 10, 2015, which are incorporated by reference herein in theirentireties.

GOVERNMENT RIGHTS STATEMENT

This invention was made with government support under 5P01CA023766 and5R21CA162002 awarded by National Institutes of Health. The governmenthas certain rights in the invention.

FIELD

Disclosed herein are methods of treating CMV (cytomegalovirus) retinitisin a human patient in need thereof, comprising administering to thehuman patient a population of allogeneic T cells comprising CMV-specificT cells, wherein the human patient is infected with HIV or has been therecipient of a solid organ transplant.

BACKGROUND

CMV Induced Retinitis: Epidemiology, Clinical Features and Morbidity

Human CMV (HCMV) is a β herpesvirus which contains a dsDNA genomeencoding >200 proteins (Chee et al., 1990, Curr Top Microbiol Immunol154:125-169). HCMV infection usually develops asymptomatic lifelonginfection in 50%-90% of healthy individuals, but can cause severeclinical complications when reactivated in immunocompromised patients(Ljungman, 2002, J Infect Dis: 186 Suppl 1:S99-S109; Fishman, 2007, NEngl J Med 357:2601-2614). One of the most serious HCMV-associateddiseases in severely immunocompromised patients is the HCMV retinitisthat leads to progressive loss of vision and blindness (Conboy et al.,1987, J Pediatr 111:343-348; Jabs et al., 1989, Ophthalmology96:1092-1099; Jacobson and Mills, 1988, Ann Intern Med 108:585-594;Egbert et al., 1980, Ann Intern Med 93: 664-670; Pollard et al., 1980,Ann Intern Med 93: 655-664).

The incidence of HCMV retinitis in AIDS patients in the United Statesbefore the advent of highly active antiretroviral therapy (HAART), wasestimated at 30% (Hoover et al., 1996, Arch Ophthalmol 114: 821-827),which has decreased to <10% in the post HAART era (Sugar et al., 2012.Am J Ophthalmol 153:1016-1024 e5; Palella et al., 1998, N Engl J Med338:853-860; Jacobson et al., 2000, Clin Infect Dis 30:231-233). Inchildren with symptomatic HCMV infection, an incidence of 5%-30% hasbeen reported (Fowler et al., 1992, N Engl J Med 326:663-667). Inseverely iatrogenically immunosuppressed adult bone marrow transplant(BMT) recipients, one study has reported to have 10 HCMV retinitis casesof 5721 during a 14-year follow-up (Crippa et al., 2001, Clin Infect Dis32:214-219). In solid organ transplant (SOT) recipients, a review ofseveral studies has reported a total of 14 cases among 12,653 patients(Egli et al., 2008, Transpl Infect Dis 10:27-43). The clinical coursefor CMV retinitis can be protracted with prolonged periods of quiescencefollowed by progression. In HIV patients prior to HAART, CMV retinitiswas associated with high rates of visual impairment (up to 98/100eye-years (EYs]) and blindness (up to 49/100 EYs) (Holbrook et al.,2003, Arch Ophthalmol 121:99-107). In the modern era, treatment withantiretroviral agents can lead to suppression of the HIV RNA circulatingin the blood (HIV load), thereby lending to immune recovery manifestedas an increase in CD4+ T cells. Despite this dramatic decrease in theincidence of CMV retinitis and the improved outcomes due to modernantiretroviral therapy, CMV retinitis and vision loss from CMV retinitiscontinue to occur (Jabs et al., 2013, Ophthalmology 120:1262-1270; Jabset al., 2010, Ophthalmology 117:2152-2161 e1-2), with the most recentlyreported rate of 0.9/100 person-year (Jabs et al., 2015, Ophthalmologypii:S0161-6420(15)00175-X, published online Apr. 16, 2015).

HCMV retinitis is diagnosed by ophthalmologic examination. Classicophthalmologic findings of HCMV retinitis include white areas of retinalnecrosis with associated hemorrhage and minimal vitreous inflammation(Lin et al., 2002, Retina 22:268-277). The current standard treatment ofCMV retinitis consists of intravenous antiviral agents such asGanciclovir and Foscarnet which are given at induction dosing for 2weeks followed by maintenance dose of oral (Valganciclovir) or IVtherapy for several weeks based on detection of CMV DNA in the blood orophthalmologic evaluation. In patients failing to respond to theseagents, cidofovir can be effective in clearing viremia or inducingregression of disease. Timely institution of treatment is critical, andin such cases approximately 50-60% of the patients will have eitherimprovement or stabilization in visual acuity, while 40% of the patientswill have progressive decline in vision (Eid et al., 2008, TransplInfect Dis 10:13-18). Responses vary between the groups of patientsdepending on the underlying disease and level of immune suppression. Thetreatment is discontinued once there is evidence of reconstitution of Tcell immunity. For patients who have ongoing immune suppression such assolid organ transplant recipients or AIDS patients with mid to higherlevel viral loads, current antiviral therapies carry significanttoxicities when administered for prolonged periods. Therefore,additional therapies are needed for patients failing to respond, or forthose who have continuous ongoing immune suppression.

Early studies in experimental mouse models provided the first evidencefor the protective effect of adoptively transferred virus specific CD8 Tcells against lethal, multiple-organ CMV infection. These studies usedas immunocompromised host, BALB/c mice treated with hematoablativetotal-body irradiation, followed by intravenous adoptive transfer ofCMV-primed CD8+ T cells and intra-plantar infection with murine CMV(MCMV) (Reddehase et al., 1988, J Virol 62:1061-1065; Reddehase et al.,1987, J Virol 61:3102-3108). Several subsequent studies defined theepitope specificities involved in protection against murine CMV and theadditive effects of adoptively transferred CD4+ T-cells.

In humans, Riddell et al. first demonstrated the efficacy of adoptivelytransferred CMV-specific CD8 T-cell clones derived from the transplantdonor prophylactically administered to recipients of BMT (bone marrowtransplant) at risk for CMV infection (Riddell et al., 1992, Science257:238-241; Walter et al., 1995, N Engl J Med 333:1038-1044). Theefficacy of donor derived CMV-specific T-cells for the treatment of CMVviremia and disease was subsequently demonstrated (Einsele et al., 2002,Blood 99:3916-3922; Feuchtinger et al., 2010, Blood 116:4360-4367;Koehne et al., 2015, Biol Blood Marrow Transplant pii:S1083-8791(15)00372-9, published online May 29, 2015; Peggs et al.,2003, Lancet 362:1375-1377). Importantly, these initial trialsdemonstrated that CMV-specific T-cells can effectively treat CNSinfections like encephalitis (Einsele et al., 2002, Blood 99:3916-3922;Feuchtinger et al., 2010, Blood 116:4360-4367), suggesting that theseT-cells can penetrate the blood brain barrier. Similarly, in thetreatment of Epstein-Barr virus related lymphoproliferative diseases(EBV-LPDs) developing in BMT recipients, it has been previously shownthat adoptively transferred transplant donor derived EBV-specificT-cells can cause complete regressions of CNS lymphomas, providingevidence that adoptively transferred T-cells can home to the CNS(central nervous system) (Doubrovina et al., 2012, Blood 119:2644-2656).

Further advancements in this field evolved to address specificlimitations of this therapy that would limit broad application of thistreatment such as; the lack of timely availability of donor derivedvirus specific T-cells and the inability to generate cells fromseronegative and cord blood donors. To overcome this limitation,pre-generated third party donor derived virus specific T-cells could bereadily available for treatment of serious viral infections in suchpatients. Several groups have demonstrated the safety and potentialefficacy of third party donor derived virus specific T cells for thetreatment of EBV (Epstein-Barr virus), CMV and adenovirus infections inBMT and SOT (solid organ transplant) recipients (Haque et al., 2007,Blood 110:1123-1131; Leen et al., 2013, Blood 121:5113-5123).

Retina as an Immune Privilege Site: Pathogenesis and Implications forTreatment

The term ‘immune-privileged site’ was created in the 1940s by Sir PeterMedawar (Medawar, 1948, Br J Exp Pathol 29:58-69). In 1977, Barker andBillingham used this term to express the exemption of sites (such as thebrain, ovary, testis, pregnant uterus, placenta, eye and the hamstercheek pouch) from immune responses (Barker and Billingham, 1977, AdvImmunol 25:1-54). Similarly, pathogen-mediated ocular inflammation canbe harmful to the eye. Since minor inflammation can result in impairedvision or even blindness, the eye is naturally designed as an immuneprivileged site where infections usually do not lead to destructiveimmune reactions (Griffith et al., 1995, Science, 270:1189-1192). Theunderlying mechanism has been hypothesized to involve Fas ligand(FasL)-mediated programmed cell death (also called apoptosis) of Fas(CD95)-expressing T cells when attracted to the infection sites(Griffith et al., 1995, Science, 270:1189-1192). In this case, activatedT cells are eliminated through ligation of Fas by FasL and no seriousimmune reactions are induced. TGF β is another cytokine present in theeye that inhibits Th1 cytokine mediated tissue destruction (Gabrielianet al., 1994, Invest Ophthalmol Vis Sci 35:4253-4259). Thus, the damageto the eye is minimized. However, CMV infection of human eyes is shownto cause large-scaled cell death and tremendous visual dysfunction (Jabset al., 1989, Ophthalmology 96:1092-1099; Jacobson and Mills, 1988, AnnIntern Med 108:585-594).

The retina is anatomically protected from invading pathogens orinflammatory cells by the inner and outer blood-retina barrier. Theinner blood-retina barrier consists of microvascular endothelial cellsand the outer blood-retina barrier consists of RPE (retinal pigmentepithelium) cells. Both cell types form functional tight junctions andare responsible for selective transport of essential molecules and forkeeping out unwanted pathogens or activated leukocytes. It has beensuggested that, in CMV infection, the internal blood-retinal barrier isdisrupted after primary CMV replication in endothelial cells, allowingCMV particles to reach retinal glial cells. Subsequently, CMV mightspread towards the RPE (Rao et al., 1998, Trans Am Ophthalmol Soc96:111-126). Although glial cells, microvascular endothelial cells andRPE cells are major targets of CMV infection in the eye, all 10 layersof the retina are sites of necrotic lesions (Rao et al., 1998, Trans AmOphthalmol Soc 96:111-126; Pecorella et al., 2000, Br J Ophthalmol84:1275-1281; Palestine et al., 1984, Ophthalmology 91:1092-1099).

These anatomic and physiological features of the retina are thought tocontribute to the characteristic features of CMV retinitis that aredistinct from CMV infections in other organs; such as the occurrence ofretinitis later in the course of infection or immunosuppression, in theabsence of CMV viremia, and as a paradoxical infection occurring despitereconstitution of CD4+ T-cell count (Song et al., 2002, Retina22:262-267). The same anatomical features can also potentially limit theefficacy of systemically administered anti-viral agents. For example,systemic CMV infections can be successfully treated with theanticytomegalovirus drugs ganciclovir, foscarnet or cidofovir, except incases infected with ganciclovir resistant CMV strains. However, in somecases of retinitis, disease progression has been observed despitecontinuous antiviral therapy and proven drug sensitivity of isolatedvirus strains, suggesting that adequate drug concentrations ofsystemically administered drugs may not be achieved in the eye.Protocols for intravitreal administration of ganciclovir and foscarnethave therefore been implemented for treatment of CMV retinitis.

T-Cell Immunity and CMV Infection

The adaptive immune system, particularly CD8+ T cells, plays a key rolein the control of acute viral infections, including CMV infection(Crough and Khanna, 2009, Clin Microbiol Rev 22:76-98, Table ofContents). Viral-specific effector CD8+ T cells exert their antiviralactivities through the production of type 1 cytokines such as interferonγ (IFN-γ) and tumor necrosis factor α (TNF-α), as well as through theirantigen specific cytolytic activity. In both mouse models and in humans,reconstitution of CMV-specific CD8+ and CD4+ T-cells is critical for thecontrol of CMV viremia and infections in recipients of both bone marrow(Quinnan et al., 1982, N Engl J Med 307:7-13; Hakki et al., 2003, Blood102:3060-3067; Podlech et al., 1998, J Gen Virol 79:2099-2104) and solidorgan transplants (Kumar et al., 2009, Am J Transplant 9:1214-1222;Pipeling et al., 2011, J Infect Dis 204:1663-1671).

CMV retinitis also occurs in patients with compromised cellularimmunity, and in a proportion of patients, development of retinitis isassociated with prior or concurrent reactivation of CMV in blood as wellas other CMV end organ disease such as colitis or pneumonitis (Eid etal., 2008, Transpl Infect Dis 10:13-18). However, the median time toonset of retinitis is 6-12 months after initiation of immunosuppressionor after transplant (Fishman, 2007, N Engl J Med 357:2601-2614; Crippaet al., 2001, Clin Infect Dis 32:214-219), which is a later time whenthe full effect of immunosuppressive medications in recipients of solidorgan transplants are well established. For HIV patients, the primaryclinical parameters determining risk for development of CMV retinitisare the post-HAART HIV viral load and CD4+ T helper cell counts ≤100/μl(Lin et al., 2002, Retina 22:268-277). CMV retinitis in HIV-positivepatients also occurs later than CMV-associated conditions in otherorgans (colon, lungs, liver) affected by CMV infection. Furthermore, theT-cell response may also contribute to ocular pathology induced by CMV,as reflected by the development of uveitis in HIV patients at the timeof CD4 T-cell recovery following institution of HAART therapy.

Taken together, these findings suggest that the rarity and lateincidence of chorioretinitis reflect the profound degree of T-celldeficiency together with the resistance created by an intactblood-retinal barrier.

Earlier studies in murine CMV retinitis models have suggested thatadoptive transfer of CMV-specific CD8+ T-cells could be protectiveagainst CMV retinitis developing in immunocompromised mice (Bigger etal., 1999, Invest Ophthalmol Vis Sci 40:2608-2613; Lu et al., 1997,Invest Ophthalmol Vis Sci 38:301-310). These studies usedimmunocompromised mice (thymectomized and T-cell depleted BALB/c mice)in whom retinitis was artificially induced by injecting infectious virusdirectly into the eye via the superciliary route, and groups of animalswere infused with murine CMV-specific T-cells or control T-cells 2 hoursprior to injection of virus. Although this model yielded the phenotypicand pathologic characteristics of CMV induced retinitis, this model ishighly non-physiological, and the results cannot be extrapolated to thehuman retinitis treatment for several reasons. (1) In contrast to themouse model in which CMV is injected directly into the eye, the intactretinal barrier is normally difficult to disrupt, and retinal human CMVinfection develops through the retinal endothelium and occurs severalmonths after immunosuppression, (2) The T-cells were transferred at thetime of introduction of the infection in the murine model, while inhuman patients developing CMV retinitis, CMV-specific T-cells would bedeficient or at least non-functional for some time (detailed above), andlastly (3) CMV is highly species specific. Early studies after isolationof murine and human cytomegaloviruses had observed that MCMV could notbe propagated in human tissue and HCMV did not replicate in murine cells(Weller, 1970, J Infect Dis 122:532-539). After further exploration, ithas been generally accepted that the cytomegaloviruses are highlyspecies specific: Each virus replicates only in cells of its own orclosely related host species. Therefore, murine CMV (MCMV) is notanalogous to human CMV. There are also significant differences in theclinical spectrum of MCMV infection in that transplacental infectiondoes not occur with MCMV, and even artificial introduction of MCMV atearly stages of embryonic development does not result in CNS infectionin mice, while human congenital CMV causes significant neurologicalsequelae. MCMV has been extensively studied and investigated for thepotential use of this virus as a stand-in for human CMV (HCMV) todevelop a mouse model, primarily for preclinical studies for vaccinestrategies against human CMV, specifically to prevent congenital CMVinfections. However, a chief limitation of the MCMV model for testingvaccines against congenital CMV infection has been the inability of thevirus to infect the fetus by transplacental route, suggesting that MCMVcauses a different spectrum of disease than HCMV. Although the precisemolecular/cellular basis for the species-specificity of CMV remainsunknown, studies by Maul et al. and others have shown that there arespecific differences in the genes encoding the immediate early 1, 2 and3 proteins of the MCMV and HCMV viruses, which have differential effectson the host cell transcriptional repressor DAxx as well histonedeacetylase, thereby affecting their ability for host infection (Mauland Negorev, 2008, Med Microbiol Immunol 197:241-249). These differencesmay also affect the host immune response to the virus, rendering thismodel less applicable to evaluation of immunotherapies for human CMV.

In order to overcome the species specificity of CMV infection,investigators have used human tissue explants; fetal thymus/liver(Mocarski et al., 1993, Proc Natl Acad Sci USA 90:104-108; Wang et al.,2005, J Virol 79:2115-2123; Brown et al., 1995, J Infect Dis171:1599-1603) or fetal retinal tissue implants (Bidanset et al., 2001,J Infect Dis 184:192-195) maintained in SCID/hu mice that could allowthe inoculation and propagation of HCMV in human cells which could thenbe used to test antiviral drugs or other therapeutic interventionsspecific for HCMV. Although these models are somewhat useful inevaluation of antiviral drugs (Kern, 2006, Antiviral Res 71:164-171),there is less information about exploiting the SCID/hu implant model forevaluation of HCMV vaccines and immunotherapies. Khanna and colleaguesstudied vaccine responses in a small animal model, in which HLA-2transgenic mice immunized with replication-deficient adenovirus vectorsexpressing HCMV epitopes were used as a way to overcome speciesspecificity of CMV viruses (Zhong et al., 2008, PLoS One 3:e3256). Thischimeric vaccine demonstrated strong HCMV-specific CD8+ and CD4+ T-cellresponses, as well as virus-neutralizing antibody. Although not a true“HCMV challenge” in a heterologous animal model, these experimentsnevertheless represent an innovative approach to overcoming the problemof species specificity in CMV vaccine models.

Thus far it is clear that CMV retinitis exclusively occurs in patientswith deficient T-cell immunity. In bone marrow transplant recipients, ithas been reported that adoptive transfer of transplant donor derived orthird party donor derived CMV specific T-cells restores T-cell immunityagainst CMV infection of the CNS such as encephalitis (Koehne et al.,2015, Biol Blood Marrow Transplant S1083-8791(15)00372-9, publishedonline May 29, 2015; Feuchtinger et al., 2010, Blood 116:4360-4367), andin some cases of CMV retinitis (Gupta et al., 2015, Ophthalmic SurgLasers Imaging Retina 46:80-82). Since third party donor derived T-cellshave a limited survival after infusion before they undergo immunerejection within the allogeneic recipient, this approach may be used forbridging anti-CMV T-cell immunity in recipients of bone marrowtransplants until reconstitution of T-cell immunity. However, there arespecific differences in host physiology and CMV infection betweenrecipients of solid organ transplant (SOT) and HIV-infected patients onone hand, and bone marrow transplant recipients on the other hand,because of which data from BMT recipients cannot be extrapolated tothese two groups of patients. First of all, in recipients of SOT, CMVreactivation and original infection occurs within the donor cells(Hammond et al., 2013, Transpl Infect Dis 15:163-170; Harvala et al.,2013, J Med Virol 85:893-898), while BMT recipients experience host CMVreactivation. The infusion of third party CMV specific T-cells in SOTwould thus carry the potential risk of precipitating immune rejection ofthe transplanted tissue carrying the CMV infection. Furthermore,independent of other factors, the inflammatory response resulting fromCMV infection in SOT recipients could also trigger a rejection episodeby enhancing antigen presentation, thus potentially placing thesepatients at high risk of organ allograft rejection by this treatmentapproach. Secondly, SOT recipients and HIV-infected patients have anindefinite period of immunodeficiency because of immunosuppressivetherapy to prevent rejection, or variable CD4 count recovery with orwithout HAART therapy, respectively. In contrast, BMT recipients have afinite period of immunodeficiency in the absence of GvHD(graft-versus-host disease). This would necessitate multiple ongoingdoses of CMV specific T-cells to treat CMV infection in SOT andHIV-infected patients, which could potentially compound the risk ofallograft rejection in SOT recipients and immune recovery uveitis inHIV-infected patients. The efficacy of T cell therapy could alsopotentially be compromised by the lack of adequate number of T-celldoses in SOT recipients and HIV-infected patients.

Therefore, while there is a need for additional therapies for thetreatment of CMV retinitis in human patients who are infected with HIVor who have been solid organ transplant recipients, these previousstudies mentioned above have limited applicability.

Citation of a reference herein shall not be construed as an admissionthat such is prior art to the present disclosure.

SUMMARY OF THE INVENTION

The present invention relates to methods of treating CMV(cytomegalovirus) retinitis in a human patient who is infected with HIV.The present invention further relates to methods of treating CMVretinitis in a human patient who has been the recipient of a solid organtransplant.

In one aspect, provided herein are methods of treating CMV retinitis ina human patient in need thereof, comprising administering to the humanpatient a population of allogeneic T cells comprising CMV-specific Tcells; wherein the human patient is infected with HIV. In variousembodiments, the human patient has AIDS (acquired immune deficiencysyndrome).

In another aspect, provided herein are methods of treating CMV retinitisin a human patient in need thereof, comprising administering to thehuman patient a population of allogeneic T cells comprising CMV-specificT cells; wherein the human patient has been the recipient of a solidorgan transplant from a transplant donor.

In various embodiments, the population of allogeneic T cells that isadministered to the human patient is restricted by an HLA allele sharedwith at least some, optionally all, of the CMV-infected cells (e.g., theinfected cells of the retina).

In certain embodiments, preferably in addition to being restricted by anHLA allele shared with at least some, optionally all, of theCMV-infected cells (e.g., the infected cells of the retina), thepopulation of allogeneic T cells comprising CMV-specific T cells sharesat least 2 out of 8 HLA alleles (e.g., two HLA-A alleles, two HLA-Balleles, two HLA-C alleles, and two HLA-DR alleles) with at least some,optionally all, of the CMV-infected cells (e.g., the infected cells ofthe retina).

In specific embodiments, the methods of treating CMV retinitis describedherein further comprise prior to the administering step a step ofascertaining at least one HLA allele of the human patient byhigh-resolution typing.

In various embodiments, the methods of treating CMV retinitis furthercomprise prior to the administering step a step of generating thepopulation of allogeneic T cells in vitro.

In certain embodiments, the step of generating the population ofallogeneic T cells in vitro comprises sensitizing (i.e., stimulating)allogeneic T cells to one or more CMV antigens so as to produceCMV-specific T cells.

In certain embodiments, the step of generating the population ofallogeneic T cells in vitro comprises sensitizing allogeneic T cellsusing dendritic cells (preferably, the dendritic cells are derived fromthe donor of allogeneic T cells). In specific embodiments, the step ofsensitizing allogeneic T cells using dendritic cells comprises loadingthe dendritic cells with at least one immunogenic peptide derived fromone or more CMV antigens. In specific embodiments, the step ofsensitizing allogeneic T cells using dendritic cells comprises loadingthe dendritic cells with a pool of overlapping peptides derived from oneor more CMV antigens.

In certain embodiments, the step of generating the population ofallogeneic T cells in vitro comprises sensitizing allogeneic T cellsusing cytokine-activated monocytes (preferably, the cytokine-activatedmonocytes are derived from the donor of allogeneic T cells). In specificembodiments, the step of sensitizing allogeneic T cells usingcytokine-activated monocytes comprises loading the cytokine-activatedmonocytes with at least one immunogenic peptide derived from one or moreCMV antigens. In specific embodiments, the step of sensitizingallogeneic T cells using cytokine-activated monocytes comprises loadingthe cytokine-activated monocytes with a pool of overlapping peptidesderived from one or more CMV antigens.

In certain embodiments, the step of generating the population ofallogeneic T cells in vitro comprises sensitizing allogeneic T cellsusing peripheral blood mononuclear cells (preferably, the peripheralblood mononuclear cells are derived from the donor of allogeneic Tcells). In specific embodiments, the step of sensitizing allogeneic Tcells using peripheral blood mononuclear cells comprises loading theperipheral blood mononuclear cells with at least one immunogenic peptidederived from one or more CMV antigens. In specific embodiments, the stepof sensitizing allogeneic T cells using peripheral blood mononuclearcells comprises loading the peripheral blood mononuclear cells with apool of overlapping peptides derived from one or more CMV antigens.

In certain embodiments, the step of generating the population ofallogeneic T cells in vitro comprises sensitizing allogeneic T cellsusing an EBV-transformed B lymphocyte cell line (EBV-BLCL). In specificembodiments, the step of sensitizing allogeneic T cells using anEBV-BLCL comprises loading the EBV-BLCL cells with at least oneimmunogenic peptide derived from one or more CMV antigens. In specificembodiments, the step of sensitizing allogeneic T cells using anEBV-BLCL comprises loading the EBV-BLCL cells with a pool of overlappingpeptides derived from one or more CMV antigens.

In certain embodiments, the step of generating the population ofallogeneic T cells in vitro comprises sensitizing allogeneic T cellsusing artificial antigen-presenting cells (AAPCs). In specificembodiments, the step of sensitizing allogeneic T cells using AAPCscomprises loading the AAPCs with at least one immunogenic peptidederived from one or more CMV antigens. In specific embodiments, the stepof sensitizing allogeneic T cells using AAPCs comprises loading theAAPCs with a pool of overlapping peptides derived from one or more CMVantigens. In specific embodiments, the step of sensitizing allogeneic Tcells using AAPCs comprises engineering the AAPCs to express at leastone immunogenic CMV peptide or protein in the AAPCs.

In a specific embodiment, the pool of overlapping peptides is a pool ofoverlapping pentadecapeptides.

In specific embodiments, the methods of treating CMV retinitis describedherein further comprise, after sensitizing, cryopreserving theallogeneic T cells.

In specific embodiments, the methods of treating CMV retinitis describedherein further comprise, before the administering step, steps of thawingcryopreserved CMV-antigen sensitized allogeneic T cells, and expandingthe allogeneic T cells in vitro, to produce the population of allogeneicT cells.

In certain embodiments, the methods of treating CMV retinitis describedherein further comprise, before the administering step, a step ofthawing a cryopreserved form of the population of allogeneic T cells.

In various embodiments, the population of allogeneic T cells is derivedfrom a T cell line. In certain embodiments, the methods of treating CMVretinitis described herein further comprise, before the administeringstep, a step of selecting the T cell line from a bank of a plurality ofcryopreserved T cell lines (preferably each comprising CMV-specific Tcells). In certain embodiments, the methods of treating CMV retinitisdescribed herein further comprise, before the administering step, a stepof thawing a cryopreserved form of the T cell line. In specificembodiments, the methods of treating CMV retinitis described hereinfurther comprises, before the administering step, a step of expandingthe T cell line (for example, after thawing a cryopreserved form of theT cell line) in vitro.

In specific embodiments, the CMV-specific T cells administered inaccordance with the methods described herein recognize CMVpp65.

In specific embodiments, the CMV-specific T cells administered inaccordance with the methods described herein recognize CMV IE1.

In specific embodiments, the population of allogeneic T cells has notbeen transduced ex vivo with a gene that encodes a CMV-specific T-cellreceptor.

In specific embodiments, at least some, optionally all, of the cells ofthe population of allogeneic T cells are rapamycin-sensitive.

In specific embodiments, the population of allogeneic T cells is notadministered in combination with a PD-1 antagonist.

In certain embodiments, the administering is by infusion of thepopulation of allogeneic T cells. In some embodiments, the infusion isbolus intravenous infusion.

In certain embodiments, the administering comprises administering atleast about 1×10⁵ T cells of the population of allogeneic T cells per kgper dose per week to the human patient. In some embodiments, theadministering comprises administering about 1×10⁶ to about 2×10⁶ T cellsof the population of allogeneic T cells per kg per dose per week to thehuman patient. In a specific embodiment, the administering comprisesadministering about 1×10⁶ T cells of the population of allogeneic Tcells per kg per dose per week to the human patient. In another specificembodiment, the administering comprises administering about 2×10⁶ Tcells of the population of allogeneic T cells per kg per dose per weekto the human patient.

In certain embodiments, the methods of treating CMV retinitis describedherein comprise administering at least 2 doses of the population ofallogeneic T cells to the human patient. In specific embodiments, themethods of treating CMV retinitis described herein compriseadministering 2, 3, 4, 5, or 6 doses of the population of allogeneic Tcells to the human patient.

In certain embodiments, the methods of treating CMV retinitis describedherein comprise administering a first cycle of one dose per week of thepopulation of allogeneic T cells for 3 consecutive weeks followed by awashout period during which no dose of the population of allogeneic Tcells is administered, followed by a second cycle of the one dose perweek of the population of allogeneic T cells for 3 consecutive weeks. Inspecific embodiments, the methods of treating CMV retinitis describedherein comprise administering two, three, four, five, or six cycles ofone dose per week of the population of allogeneic T cells for 3consecutive weeks, each cycle separated by a washout period during whichno dose of the population of allogeneic T cells is administered. In aspecific embodiment, the washout period is about three weeks.

In certain embodiments, the methods of treating CMV retinitis furthercomprise, after administering to the human patient the population ofallogeneic T cells, administering to the human patient a secondpopulation of allogeneic T cells comprising CMV-specific T cells;wherein the second population of allogeneic T cells is restricted by adifferent HLA allele shared with at least some, optionally all, of theCMV-infected cells. In a specific embodiment, the methods of treatingCMV retinitis comprise administering a first cycle of one dose per weekof the population of allogeneic T cells for 3 consecutive weeks followedby a washout period during which no dose of the population of allogeneicT cells is administered, followed by a second cycle of one dose per weekof the second population of allogeneic T cells for 3 consecutive weeks.In a further specific embodiment, the washout period is about threeweeks. In certain embodiments, the human patient has no response, anincomplete response, or a suboptimal response (i.e., the human patientmay still have a substantial benefit from continuing treatment, but hasreduced chances of optimal long-term outcomes) after administering thepopulation of allogeneic T cells and prior to administering the secondpopulation of allogeneic T cells.

In specific embodiments, the human patient has an active, not latent,CMV infection.

In specific embodiments, a CMV in the human patient has at least onemutation in its genome that confers resistance to one or more anti-viralagents. In a specific embodiment, the one or more anti-viral agents areselected from the group consisting of ganciclovir, foscarnet,valganciclovir, cidofovir, leflunomide, and combinations thereof. In aspecific embodiment, the mutation is in the UL97 gene. In anotherspecific embodiment, the mutation is in the UL54 gene. In anotherspecific embodiment, a first mutation is in the UL97 gene and a secondmutation is in the UL54 gene.

In certain embodiments, the human patient has been the recipient of asolid organ transplant from a transplant donor. In specific embodiments,the solid organ transplant that the human patient has received is akidney transplant, a liver transplant, a heart transplant, an intestinaltransplant, a pancreas transplant, a lung transplant, a small boweltransplant, or a combination thereof. In a specific embodiment, thesolid organ transplant that the human patient has received is a kidneytransplant. In specific embodiments wherein the human patient has beenthe recipient of a solid organ transplant from a transplant donor, thepopulation of allogeneic T cells is derived from a donor other than thetransplant donor.

In specific embodiments, the human patient has not been the recipient ofa hematopoietic stem cell transplant (e.g., a bone marrow transplant, aperipheral blood stem cell transplant, or a cord blood transplant).

In specific embodiments, the human patient has failed a previous therapyto treat the CMV retinitis. In a specific embodiment, the CMV retinitisis resistant to the previous therapy. In a specific embodiment, thehuman patient has been taken off the previous therapy due to intoleranceof the therapy. In specific embodiments, the previous therapy istreatment with at least one anti-viral agent. In a specific embodiment,the at least one anti-viral agent is selected from the group consistingof ganciclovir, foscarnet, valganciclovir, cidofovir, leflunomide, andcombinations thereof.

In specific embodiments, the methods of treating CMV retinitis furthercomprise concurrently treating the human patient with an anti-viralcompound to treat the CMV retinitis. In a specific embodiment, theanti-viral compound is selected from the group consisting ofganciclovir, foscarnet, valganciclovir, cidofovir, leflunomide, andcombinations thereof.

In specific embodiments, the methods of treating CMV retinitis furthercomprise prior to said administering step a step of genotyping a CMV ofthe human patient

BRIEF DESCRIPTION OF FIGURES

FIG. 1 depicts the schema of treating CMV retinitis using CMV-specific Tcells.

FIG. 2 is a summary of patients treated with third party donor-derivedCMV-specific T cells.

FIG. 3 depicts the clinical chronology of patient #5. CMV viral titer ispresented in international units per ml (IU/ml) (1 IU=0.53 copies ofCMV).

FIG. 4 shows the ophthalmologic examination results and Fundusphotography of patient #5 before and after T cell therapy. VA: visualacuity. CF: count fingers. OD: oculus dexter, i.e., right eye. OS:oculus sinister, i.e., left eye.

FIG. 5 shows a representative example (patient #5) illustrating thatincreases in CMV-specific T cells were associated with decline in CMVviral load in blood.

DETAILED DESCRIPTION

The present invention relates to methods of treating CMV(cytomegalovirus) retinitis in a human patient who is infected with HIV.The present invention further relates to methods of treating CMVretinitis in a human patient who has been the recipient of a solid organtransplant. The invention provides a T cell therapy method that iseffective in treating CMV retinitis in a human patient with low or notoxicity, wherein the human patient is infected with HIV or has been therecipient of a solid organ transplant.

In one aspect, provided herein are methods of treating CMV retinitis ina human patient in need thereof, comprising administering to the humanpatient a population of allogeneic T cells comprising CMV-specific Tcells; wherein the human patient is infected with HIV. In variousembodiments, the human patient has AIDS (acquired immune deficiencysyndrome).

In another aspect, provided herein are methods of treating CMV retinitisin a human patient in need thereof, comprising administering to thehuman patient a population of allogeneic T cells comprising CMV-specificT cells; wherein the human patient has been the recipient of a solidorgan transplant from a transplant donor.

5.1. A Population of Allogeneic T Cells Restricted by an Shared HLAAllele with the Infected Cells of CMV Retinitis

According to the invention, a population of allogeneic T cellscomprising CMV-specific T cells is administered to the human patient. Ina specific embodiment, the population of allogeneic T cells that isadministered to the human patient is restricted by an HLA allele sharedwith at least some, optionally all, of the CMV-infected cells (e.g., theinfected cells of the retina). Preferably, the population of allogeneicT cells has demonstrated anti-CMV cytotoxic activity, measured by amethod known in the art (for example, as described in Trivedi et al.,2005, Blood 105:2793-2801; or Hasan et al., 2009, J Immunol 183:2837-2850).

When the human patient is infected with HIV, the CMV-infected cells havethe same HLA type (i.e., assignment) as the human patient. In specificembodiments when the human patient is infected with HIV, the populationof allogeneic T cells that is administered to the human patient isrestricted by an HLA allele shared with all of the CMV-infected cells.In some embodiments, this HLA allele restriction is ensured byascertaining the HLA assignment of the CMV-infected cells, and selectinga population of allogeneic T cells comprising CMV-specific T cells (or aT cell line from which to derive the population of allogeneic T cells)restricted by an HLA allele of such CMV-infected cells. In otherembodiments, when ascertaining the HLA assignment of the CMV-infectedcells is not possible (or is possible but not performed), this HLAallele restriction is ensured by ascertaining the HLA assignment of thehuman patient (e.g., by using non-infected cells or tissue from thehuman patient), and selecting a population of allogeneic T cellscomprising CMV-specific T cells (or a T cell line from which to derivethe population of allogeneic T cells) restricted by an HLA allele of thehuman patient.

When the human patient has been the recipient of a solid organtransplant from a transplant donor, the CMV-infected cells in most casescontain both infected cells of the patient origin and infected cells ofthe transplant donor origin. In specific embodiments when the humanpatient has been the recipient of a solid organ transplant, thepopulation of allogeneic T cells that is administered to the humanpatient can be restricted by an HLA allele shared with all of theCMV-infected cells. In specific embodiments when the human patient hasbeen the recipient of a solid organ transplant, the population ofallogeneic T cells that is administered to the human patient can berestricted by an HLA allele shared with at least some of theCMV-infected cells. In some embodiments, a population of allogeneic Tcells comprising CMV-specific T cells (or a T cell line from which toderive the population of allogeneic T cells) restricted by an HLA alleleshared by both the human patient and the transplant donor is selectedfor administering (the population of allogeneic T cells that isadministered to the human patient is then restricted by an HLA alleleshared with all of the CMV-infected cells). In a specific embodimentwhen the human patient is at a high risk for organ allograft rejection,and CMV retinitis is the main problem without CMV viremia, a populationof allogeneic T cells comprising CMV-specific T cells (or a T cell linefrom which to derive the population of allogeneic T cells) restricted byan HLA allele of the human patient can be selected for administering(the population of allogeneic T cells that is administered to the humanpatient is then restricted by an HLA allele shared with at least some ofthe CMV-infected cells). In a specific embodiment (e.g., when the originof the CMV-infected cells is determined to be the human patient only), apopulation of allogeneic T cells comprising CMV-specific T cells (or a Tcell line from which to derive the population of allogeneic T cells)restricted by an HLA allele of the human patient is selected foradministering. In a specific embodiment (e.g., when the origin of theCMV-infected cells is determined to be the transplant donor only), apopulation of allogeneic T cells comprising CMV-specific T cells (or a Tcell line from which to derive the population of allogeneic T cells)restricted by an HLA allele of the transplant donor is selected foradministering. In some embodiments wherein the patient has CMV viremia,a population of allogeneic T cells comprising CMV-specific T cells (or aT cell line from which to derive the population of allogeneic T cells)that is restricted by an HLA allele shared by both the human patient andthe transplant donor can be selected for administering. In certainembodiments, the methods further comprise prior to the administeringstep, a step of ascertaining the HLA assignment of the CMV-infectedcells, the human patient, the transplant donor, or both the humanpatient and the transplant donor (as the case may be).

The origin of the CMV-infected cells can be determined by any methodknown in the art, for example, by analyzing variable tandem repeats(VTRs) (which is a method that uses unique DNA signature of small DNAsequences of different people to distinguish between the recipient andthe donor of a transplant), or by looking for the presence or absence ofchromosome Y if the donor and the recipient of a transplant are ofdifferent sexes (which is done by cytogenetics or by FISH (fluorescencein situ hybridization)).

In some embodiments of ascertaining an HLA assignment, at least 4 HLAloci (preferably HLA-A, HLA-B, HLA-C, and HLA-DR) are typed. In someembodiments of ascertaining an HLA assignment, 4 HLA loci (preferablyHLA-A, HLA-B, HLA-C, and HLA-DR) are typed. In some embodiments ofascertaining an HLA assignment, 6 HLA loci are typed. In someembodiments of ascertaining an HLA assignment, 8 HLA loci are typed.

In certain embodiments, preferably in addition to being restricted by anHLA allele shared with at least some, optionally all, of theCMV-infected cells (e.g., the infected cells of the retina), thepopulation of allogeneic T cells comprising CMV-specific T cells sharesat least 2 HLA alleles with at least some, optionally all, of theCMV-infected cells (e.g., the infected cells of the retina). Preferably,the population of allogeneic T cells has demonstrated anti-CMV cytotoxicactivity, measured by a method known in the art (for example, asdescribed in Trivedi et al., 2005, Blood 105:2793-2801; or Hasan et al.,2009, J Immunol 183: 2837-2850). In specific embodiments, the populationof allogeneic T cells comprising CMV-specific T cells shares at least 2out of 8 HLA alleles (for example, two HLA-A alleles, two HLA-B alleles,two HLA-C alleles, and two HLA-DR alleles) with at least some,optionally all, of the CMV-infected cells.

When the human patient is infected with HIV, the CMV-infected cells havethe same HLA type (i.e., assignment) as the human patient. In specificembodiments when the human patient is infected with HIV, the populationof allogeneic T cells that is administered to the human patient sharesat least 2 HLA alleles with all of the CMV-infected cells. In someembodiments, this sharing is ensured by ascertaining the HLA assignmentof the CMV-infected cells, and selecting a population of allogeneic Tcells comprising CMV-specific T cells (or a T cell line from which toderive the population of allogeneic T cells) that shares at least 2(e.g., at least 2 out of 8) HLA alleles with such CMV-infected cells. Inother embodiments, when ascertaining the HLA assignment of theCMV-infected cells is not possible (or is possible but not performed),this sharing is ensured by ascertaining the HLA assignment of the humanpatient (e.g., by using non-infected cells or tissue from the humanpatient), and selecting a population of allogeneic T cells comprisingCMV-specific T cells (or a T cell line from which to derive thepopulation of allogeneic T cells) that shares at least 2 (e.g., at least2 out of 8) HLA alleles with the human patient.

When the human patient has been the recipient of a solid organtransplant from a transplant donor, the CMV-infected cells in most casescontain both infected cells of the patient origin and infected cells ofthe transplant donor origin. In specific embodiments when the humanpatient has been the recipient of a solid organ transplant, thepopulation of allogeneic T cells that is administered to the humanpatient can share at least 2 (e.g., at least 2 out of 8) HLA alleleswith all of the CMV-infected cells. In specific embodiments when thehuman patient has been the recipient of a solid organ transplant, thepopulation of allogeneic T cells that is administered to the humanpatient can share at least 2 (e.g., at least 2 out of 8) HLA alleleswith at least some of the CMV-infected cells. In some embodiments, apopulation of allogeneic T cells comprising CMV-specific T cells (or a Tcell line from which to derive the population of allogeneic T cells)that shares at least 2 (e.g., at least 2 out of 8) HLA alleles with boththe human patient and the transplant donor is selected for administering(the population of allogeneic T cells that is administered to the humanpatient then shares at least 2 (e.g., at least 2 out of 8) HLA alleleswith all of the CMV-infected cells). In a specific embodiment when thehuman patient is at a high risk for organ allograft rejection, and CMVretinitis is the main problem without CMV viremia, a population ofallogeneic T cells comprising CMV-specific T cells (or a T cell linefrom which to derive the population of allogeneic T cells) that sharesat least 2 (e.g., at least 2 out of 8) HLA alleles with the humanpatient can be selected for administering (the population of allogeneicT cells that is administered to the human patient then shares at least 2(e.g., at least 2 out of 8) HLA alleles with at least some of theCMV-infected cells). In a specific embodiment, (e.g., when the origin ofthe CMV-infected cells is determined to be the human patient only), apopulation of allogeneic T cells comprising CMV-specific T cells (or a Tcell line from which to derive the population of allogeneic T cells)that shares at least 2 (e.g., at least 2 out of 8) HLA alleles with thehuman patient is selected for administering. In a specific embodiment(e.g., when the origin of the CMV-infected cells is determined to be thetransplant donor only), a population of allogeneic T cells comprisingCMV-specific T cells (or a T cell line from which to derive thepopulation of allogeneic T cells) that shares at least 2 (e.g., at least2 out of 8) HLA alleles with the transplant donor is selected foradministering. In some embodiments when the patient has CMV-relatedviremia, a population of allogeneic T cells comprising CMV-specific Tcells (or a T cell line from which to derive the population ofallogeneic T cells) that shares at least 2 (e.g., at least 2 out of 8)HLA alleles with both the human patient and the transplant donor can beselected for administering. In certain embodiments, the methods furthercomprise prior to the administering step, a step of ascertaining the HLAassignment of the CMV-infected cells, the human patient, the transplantdonor, or both the human patient and the transplant donor (as the casemay be).

The HLA assignment (i.e., the HLA loci type) can be ascertained (i.e.,typed) by any method known in the art. Non-limiting exemplary methodsfor ascertaining the HLA assignment can be found in ASHI LaboratoryManual, Edition 4.2 (2003), American Society for Histocompatibility andImmunogenetics; ASHI Laboratory Manual, Supplements 1 (2006) and 2(2007), American Society for Histocompatibility and Immunogenetics;Hurley, “DNA-based typing of HLA for transplantation.” in Leffell etal., eds., 1997, Handbook of Human Immunology, Boca Raton: CRC Press;Dunn, 2011, Int J Immunogenet 38:463-473; Erlich, 2012, Tissue Antigens,80:1-11; Bontadini, 2012, Methods, 56:471-476; and Lange et al., 2014,BMC Genomics 15: 63.

In general, high-resolution typing is preferable for HLA typing. Thehigh-resolution typing can be performed by any method known in the art,for example, as described in ASHI Laboratory Manual, Edition 4.2 (2003),American Society for Histocompatibility and Immunogenetics; ASHILaboratory Manual, Supplements 1 (2006) and 2 (2007), American Societyfor Histocompatibility and Immunogenetics; Flomenberg et al., Blood,104:1923-1930; Kögler et al., 2005, Bone Marrow Transplant,36:1033-1041; Lee et al., 2007, Blood 110:4576-4583; Erlich, 2012,Tissue Antigens, 80:1-11; Lank et al., 2012, BMC Genomics 13:378; orGabriel et al., 2014, Tissue Antigens, 83:65-75. In specificembodiments, the methods of treating CMV retinitis described hereinfurther comprise prior to the administering step a step of ascertainingat least one HLA allele of the CMV-infected cells by high-resolutiontyping. In specific embodiments, the methods of treating CMV retinitisdescribed herein further comprise prior to the administering step a stepof ascertaining at least one HLA allele of the human patient byhigh-resolution typing. In specific embodiments when the human patienthas been the recipient of a solid organ transplant from a transplantdonor, the methods of treating CMV retinitis described herein furthercomprise prior to the administering step a step of ascertaining at leastone HLA allele of the transplant donor by high-resolution typing. Inspecific embodiments, when the human patient has been the recipient of asolid organ transplant from a transplant donor, the methods of treatingCMV retinitis described herein further comprise prior to theadministering step a step of ascertaining at least one HLA allele of thehuman patient and at least one HLA allele of the transplant donor byhigh-resolution typing.

The HLA allele by which the population of allogeneic T cells isrestricted can be determined by any method known in the art, forexample, as described in Trivedi et al., 2005, Blood 105:2793-2801;Barker et al., 2010, Blood 116:5045-5049; Hasan et al., 2009, J Immunol,183:2837-2850; or Doubrovina et al., 2012, Blood 120:1633-1646.

Preferably, the HLA allele by which the population of allogeneic T cellsis restricted and is shared with at least some, optionally all, of theCMV-infected cells is defined by high-resolution typing. Preferably, theHLA alleles that are shared between the population of allogeneic T cellsand at least some, optionally all, of the CMV-infected cells are definedby high-resolution typing. Most preferably, both the HLA allele by whichthe population of allogeneic T cells is restricted and is shared with atleast some, and optionally all, of the CMV-infected cells, and the HLAalleles that are shared between the population of allogeneic T cells andat least some, optionally all, of the CMV-infected cells are defined byhigh-resolution typing.

5.2. Obtaining or Generating a Population of Allogeneic T CellsComprising CMV-Specific T Cells

The population of allogeneic T cells comprising CMV-specific T cellsthat is administered to the human patient can be generated by a methodknown in the art, or can be selected from a preexisting bank(collection) of cryopreserved T cell lines (each T cell line comprisingCMV-specific T cells) generated by a method known in the art, and thawedand preferably expanded prior to administration. Preferably, uniqueidentifier for each T cell line in the bank is associated withinformation as to which HLA allele(s) the respective T cell line isrestricted, the HLA assignment of the respective T cell line, and/or theanti-CMV cytotoxic activity of the respective T cell line measured by amethod known in the art (for example, as described in Trivedi et al.,2005, Blood 105:2793-2801; or Hasan et al., 2009, J Immunol 183:2837-2850). The population of allogeneic T cells and the T cell lines inthe bank are preferably obtained or generated by methods describedbelow.

In various embodiments, the methods of treating CMV retinitis furthercomprise prior to the administering step a step of obtaining thepopulation of allogeneic T cells. Preferably, the population ofallogeneic T cells comprising CMV-specific T cells are derived from CMVseropositive donors.

In specific embodiments, the step of obtaining the population ofallogeneic T cells comprises fluorescence activated cell sorting forCMV-positive T cells from a population of blood cells. In a specificembodiment, the population of blood cells are peripheral bloodmononuclear cells (PBMCs) isolated from a blood sample(s) obtained froma human donor. The fluorescence activated cell sorting can be performedby any method known in the art, which normally involves staining thepopulation of blood cells with an antibody that recognizes at least oneCMV antigen before the sorting step.

In specific embodiments, the step of obtaining the population ofallogeneic T cells comprises generating the population of allogeneic Tcells in vitro. The population of allogeneic T cells can be generated invitro by any method known in the art. Non-limiting exemplary methods ofgenerating the population of allogeneic T cells can be found in Trivediet al., 2005, Blood 105:2793-2801; Hasan et al., 2009, J Immunol 183:2837-2850; Koehne et al., 2015, Biol Blood Marrow TransplantS1083-8791(15)00372-9, published online May 29, 2015; O'Reilly et al.,2007, Immunol Res 38:237-250; and O' Reilly et al., 2011, Best Practice& Research Clinical Haematology 24:381-391.

In certain embodiments, the step of generating the population ofallogeneic T cells in vitro comprises sensitizing (i.e., stimulating)allogeneic T cells to one or more CMV antigens so as to produceCMV-specific T cells. In specific embodiments, the step of generatingthe population of allogeneic T cells in vitro comprises sensitizingallogeneic T cells to one or more CMV antigens presented by antigenpresenting cells. The allogeneic T cells that are used for generatingthe population of allogeneic T cells in vitro can be isolated from thedonor of the allogeneic T cells by any method known in the art, forexample, as described in Trivedi et al., 2005, Blood 105:2793-2801;Hasan et al., 2009, J Immunol 183: 2837-2850; or O'Reilly et al., 2007,Immunol Res. 38:237-250. In a specific embodiment, the allogeneic Tcells are enriched from peripheral blood lymphocytes separated fromPBMCs of the donor of the allogeneic T cells. In a further specificembodiment, T cells are enriched from peripheral blood lymphocytesseparated from PBMCs of the donor of the allogeneic T cells by depletionof adherent monocytes followed by depletion of natural killer cells. Invarious embodiments, the allogeneic T cells are cryopreserved forstorage. In a specific embodiment, wherein the allogeneic T cells arecryopreserved, the cryopreserved allogeneic T cells are thawed andexpanded in vitro before sensitizing. In a specific embodiment, whereinthe allogeneic T cells are cryopreserved, the cryopreserved allogeneic Tcells are thawed and then sensitized, but not expanded in vitro beforesensitizing, and then optionally expanded. In specific embodiments, theallogeneic T cells are cryopreserved after sensitizing (sensitizingproduces the CMV-specific T cells). In a specific embodiment, whereinthe allogeneic T cells are cryopreserved after sensitizing, thecryopreserved allogeneic T cells are thawed and expanded in vitro toproduce the population of allogeneic T cells comprising CMV-specific Tcells. In another specific embodiment, wherein the allogeneic T cellsare cryopreserved after sensitizing, the cryopreserved allogeneic Tcells are thawed but not expanded in vitro to produce the population ofallogeneic T cells comprising CMV-specific T cells. In other variousembodiments, the allogeneic T cells are not cryopreserved. In a specificembodiment, wherein the allogeneic T cells are not cryopreserved, theallogeneic T cells are expanded in vitro before sensitizing. In aspecific embodiment, wherein the allogeneic T cells are notcryopreserved, the allogeneic T cells are not expanded in vitro beforesensitizing. In specific embodiments, the step of generating thepopulation of allogeneic T cells in vitro further comprises, aftersensitizing, cryopreserving the allogeneic T cells.

In specific embodiments, the methods of treating CMV retinitis describedherein further comprise, before the administering step, steps of thawingcryopreserved CMV-antigen sensitized allogeneic T cells, and expandingthe allogeneic T cells in vitro, to produce the population of allogeneicT cells.

In certain embodiments, the step of generating the population ofallogeneic T cells in vitro comprises sensitizing allogeneic T cellsusing dendritic cells (preferably, the dendritic cells are derived fromthe donor of allogeneic T cells). In specific embodiments, the step ofsensitizing allogeneic T cells using dendritic cells comprises loadingthe dendritic cells with at least one immunogenic peptide derived fromone or more CMV antigens. In specific embodiments, the step ofsensitizing allogeneic T cells using dendritic cells comprises loadingthe dendritic cells with a pool of overlapping peptides derived from oneor more CMV antigens.

In certain embodiments, the step of generating the population ofallogeneic T cells in vitro comprises sensitizing allogeneic T cellsusing cytokine-activated monocytes (preferably, the cytokine-activatedmonocytes are derived from the donor of allogeneic T cells). In specificembodiments, the step of sensitizing allogeneic T cells usingcytokine-activated monocytes comprises loading the cytokine-activatedmonocytes with at least one immunogenic peptide derived from one or moreCMV antigens. In specific embodiments, the step of sensitizingallogeneic T cells using cytokine-activated monocytes comprises loadingthe cytokine-activated monocytes with a pool of overlapping peptidesderived from one or more CMV antigens.

In certain embodiments, the step of generating the population ofallogeneic T cells in vitro comprises sensitizing allogeneic T cellsusing peripheral blood mononuclear cells (preferably, the peripheralblood mononuclear cells are derived from the donor of allogeneic Tcells). In specific embodiments, the step of sensitizing allogeneic Tcells using peripheral blood mononuclear cells comprises loading theperipheral blood mononuclear cells with at least one immunogenic peptidederived from one or more CMV antigens. In specific embodiments, the stepof sensitizing allogeneic T cells using peripheral blood mononuclearcells comprises loading the peripheral blood mononuclear cells with apool of overlapping peptides derived from one or more CMV antigens.

In certain embodiments, the step of generating the population ofallogeneic T cells in vitro comprises sensitizing allogeneic T cellsusing an EBV-transformed B lymphocyte cell line (EBV-BLCL), for example,an EBV strain B95.8-transformed B lymphocyte cell line (preferably, theEBV-BLCL is derived from the donor of allogeneic T cells). The EBV-BLCLcan be generated by any method known in the art, or as previouslydescribed in Trivedi et al., 2005, Blood 105:2793-2801 or Hasan et al.,2009, J Immunol 183:2837-2850. In specific embodiments, the step ofsensitizing allogeneic T cells using an EBV-BLCL comprises loading theEBV-BLCL cells with at least one immunogenic peptide derived from one ormore CMV antigens. In specific embodiments, the step of sensitizingallogeneic T cells using an EBV-BLCL comprises loading the EBV-BLCLcells with a pool of overlapping peptides derived from one or more CMVantigens.

In certain embodiments, the step of generating the population ofallogeneic T cells in vitro comprises sensitizing allogeneic T cellsusing artificial antigen-presenting cells (AAPCs). In specificembodiments, the step of sensitizing allogeneic T cells using AAPCscomprises loading the AAPCs with at least one immunogenic peptidederived from one or more CMV antigens. In specific embodiments, the stepof sensitizing allogeneic T cells using AAPCs comprises loading theAAPCs with a pool of overlapping peptides derived from one or more CMVantigens. In specific embodiments, the step of sensitizing allogeneic Tcells using AAPCs comprises engineering the AAPCs to express at leastone immunogenic CMV peptide or protein in the AAPCs.

In various embodiments, the pool of peptides is a pool of overlappingpeptides spanning an antigen of CMV. In various embodiments, the pool ofpeptides is a pool of overlapping peptides spanning more than oneantigen of CMV. In a specific embodiment, the pool of overlappingpeptides is a pool of overlapping pentadecapeptides.

In specific embodiments, the population of allogeneic T cells has beencryopreserved for storage before administering. In specific embodiments,the population of allogeneic T cells has not been cryopreserved forstorage before administering. In certain embodiments, the methods oftreating CMV retinitis described herein further comprise, before theadministering step, a step of thawing a cryopreserved form of thepopulation of allogeneic T cells.

In various embodiments, the population of allogeneic T cells is derivedfrom a T cell line. In specific embodiments, the T cell line has beencryopreserved for storage before administering. In specific embodiments,the T cell line has not been cryopreserved for storage beforeadministering. In some embodiments, the T cell line has been expanded invitro to derive the population of allogeneic T cells. In otherembodiments, the T cell line has not been expanded in vitro to derivethe population of allogeneic T cells. The T cell line can be sensitizedto one or more CMV antigens (so as to produce CMV-specific T cells, forexample, by a sensitizing step described above) before or aftercryopreservation (if the T cell line has been cryopreserved), and beforeor after expanding in vitro (if the T cell line has been expanded invitro). In certain embodiments, the methods of treating CMV retinitisdescribed herein further comprise, before the administering step, a stepof selecting the T cell line from a bank of a plurality of cryopreservedT cell lines (preferably each comprising CMV-specific T cells).Preferably, unique identifier for each T cell line in the bank isassociated with information as to which HLA allele(s) the respective Tcell line is restricted, and optionally also information as to the HLAassignment of the respective T cell line. In certain embodiments, themethods of treating CMV retinitis described herein further comprise,before the administering step, a step of thawing a cryopreserved form ofthe T cell line. In specific embodiments, the methods of treating CMVretinitis described herein further comprises, before the administeringstep, a step of expanding the T cell line (for example, after thawing acryopreserved form of the T cell line) in vitro. The T cell line and theplurality of cryopreserved T cell lines can be generated by any methodknown in the art, for example, as described in Trivedi et al., 2005,Blood 105:2793-2801; Hasan et al., 2009, J Immunol 183: 2837-2850;Koehne et al., 2015, Biol Blood Marrow Transplant S1083-8791(15)00372-9,published online May 29, 2015; O'Reilly et al., 2007, Immunol Res38:237-250; or O' Reilly et al., 2011, Best Practice & Research ClinicalHaematology 24:381-391, or as describe above for generating thepopulation of allogeneic T cells in vitro.

The population of allogeneic T cells comprising CMV-specific T cellsthat is administered to the human patient comprises CD8+ T cells, and ina specific embodiment also comprises CD4+ T cells.

The CMV-specific T cells administered in accordance with the methodsdescribed herein recognize at least one antigen of CMV. In specificembodiments, the CMV-specific T cells administered in accordance withthe methods described herein recognize CMVpp65. In specific embodiments,the CMV-specific T cells administered in accordance with the methodsdescribed herein recognize CMV IE1.

In specific embodiments, the population of allogeneic T cells has notbeen transduced ex vivo with a gene that encodes a CMV-specific T-cellreceptor.

In specific embodiments, at least some, optionally all, of the cells ofthe population of allogeneic T cells are rapamycin-sensitive.

In specific embodiments, the population of allogeneic T cells is notadministered in combination with a PD-1 antagonist.

5.3. Administration and Dosage

The route of administration of the population of allogeneic T cells andthe amount to be administered to the human patient can be determinedbased on the condition of the human patient and the knowledge of thephysician. Generally, the administration is intravenous.

In certain embodiments, the administering is by infusion of thepopulation of allogeneic T cells. In some embodiments, the infusion isbolus intravenous infusion. In certain embodiments, the administeringcomprises administering at least about 1×10⁵ T cells of the populationof allogeneic T cells per kg per dose per week to the human patient. Insome embodiments, the administering comprises administering about 1×10⁶to about 2×10⁶ T cells of the population of allogeneic T cells per kgper dose per week to the human patient. In a specific embodiment, theadministering comprises administering about 1×10⁶ T cells of thepopulation of allogeneic T cells per kg per dose per week to the humanpatient. In another specific embodiment, the administering comprisesadministering about 2×10⁶ T cells of the population of allogeneic Tcells per kg per dose per week to the human patient.

In certain embodiments, the methods of treating CMV retinitis describedherein comprise administering at least 2 doses of the population ofallogeneic T cells to the human patient. In specific embodiments, themethods of treating CMV retinitis described herein compriseadministering 2, 3, 4, 5, or 6 doses of the population of allogeneic Tcells to the human patient.

In certain embodiments, the methods of treating CMV retinitis describedherein comprise administering a first cycle of one dose per week of thepopulation of allogeneic T cells for 3 consecutive weeks followed by awashout period during which no dose of the population of allogeneic Tcells is administered, followed by a second cycle of the one dose perweek of the population of allogeneic T cells for 3 consecutive weeks. Incertain embodiments, the methods of treating CMV retinitis describedherein comprise administering at least two cycles of one dose per weekof the population of allogeneic T cells for 3 consecutive weeks, eachcycle separated by a washout period during which no dose of thepopulation of allogeneic T cells is administered. In specificembodiments, the methods of treating CMV retinitis described hereincomprise administering two, three, four, five, or six cycles of one doseper week of the population of allogeneic T cells for 3 consecutiveweeks, each cycle separated by a washout period during which no dose ofthe population of allogeneic T cells is administered. In a specificembodiment, the washout period is about three weeks. Preferably, anadditional cycle is administered only when the previous cycle has notexhibited toxicity (for example, no grade 3-5 serious adverse events,graded according to NCI CTCAE 4.0).

In a specific embodiment, the methods of treating CMV retinitisdescribed herein comprises administering a first cycle of one dose perweek of the population of allogeneic T cells for 3 consecutive weeksfollowed by a washout period during which no dose of the population ofallogeneic T cells is administered, followed by a second cycle of onedose per week of the population of allogeneic T cells for 3 consecutiveweeks, wherein each dose is about 1×10⁶ T cells of the population ofallogeneic T cells per kg, and the washout period is about three weeks.

In a specific embodiment, the methods of treating CMV retinitisdescribed herein comprises administering about 1×10⁶ T cells of thepopulation of allogeneic T cells per kg per dose per week to the humanpatient for three consecutive weeks (i.e., 3 doses).

In certain embodiments, a first dosage regimen described herein iscarried out for a first period of time, followed by a second anddifferent dosage regimen described herein that is carried out for asecond period of time, wherein the first period of time and the secondperiod of time are optionally separated by a washout period (forexample, about three weeks). Preferably, the second dosage regimen iscarried out only when the first dosage regimen has not exhibitedtoxicity (for example, no grade 3-5 serious adverse events, gradedaccording to NCI CTCAE 4.0).

The term “about” shall be construed so as to allow normal variation.

5.4. Serial Treatment with Different T Cell Populations

In certain embodiments, the methods of treating CMV retinitis furthercomprise, after administering to the human patient the population ofallogeneic T cells, administering to the human patient a secondpopulation of allogeneic T cells comprising CMV-specific T cells;wherein the second population of allogeneic T cells is restricted by adifferent HLA allele shared with at least some, optionally all, of theCMV-infected cells. The second population of allogeneic T cells can beadministered by any route and any dosage/administration regimen asdescribed in Section 4.4. In a specific embodiment, the methods oftreating CMV retinitis comprise administering a first cycle of one doseper week of the population of allogeneic T cells for 3 consecutive weeksfollowed by a washout period during which no dose of the population ofallogeneic T cells is administered, followed by a second cycle of onedose per week of the second population of allogeneic T cells for 3consecutive weeks. In a further specific embodiment, the washout periodis about three weeks.

In certain embodiments, the human patient has no response, an incompleteresponse, or a suboptimal response (i.e., the human patient may stillhave a substantial benefit from continuing treatment, but has reducedchances of optimal long-term outcomes) after administering thepopulation of allogeneic T cells and prior to administering the secondpopulation of allogeneic T cells.

In specific embodiments, two populations of allogeneic CMV-specific Tcells that are each restricted by a different HLA allele shared with atleast some, optionally all, of the CMV-infected cells are administeredserially. In specific embodiments, three populations of allogeneicCMV-specific T cells that are each restricted by a different HLA alleleshared with at least some, optionally all, of the CMV-infected cells areadministered serially. In specific embodiments, four populations ofallogeneic CMV-specific T cells that are each restricted by a differentHLA allele shared with at least some, optionally all, of theCMV-infected cells are administered serially. In specific embodiments,more than four populations of allogeneic CMV-specific T cells that areeach restricted by a different HLA allele shared with at least some,optionally all, of the CMV-infected cells are administered serially.

5.5. Combination Therapy

In various embodiments, the methods of treating CMV retinitis furthercomprise treating the human patient with an anti-viral compound to treatthe CMV retinitis. In specific embodiments, the methods of treating CMVretinitis further comprise concurrently treating the human patient withan anti-viral compound to treat the CMV retinitis. In specificembodiments, the anti-viral compound is selected from the groupconsisting of ganciclovir, foscarnet, valganciclovir, cidofovir,leflunomide, and combinations thereof.

The anti-viral compound administered to the human patient may beadministered to the human patient by a variety of routes. These include,but are not limited to, intravitreal, parenteral, intranasal,intratracheal, oral, intradermal, topical, intramuscular,intraperitoneal, transdermal, intravenous, infusion, intratumoral,conjunctival, subcutaneous, pulmonary, and any other local or systemicroutes.

The amount of the anti-viral compound described herein or apharmaceutical composition thereof which will be effective in thetreatment of the CMV retinitis will depend on the nature of the diseaseand the condition of the patient, and can be determined by standardclinical techniques and the knowledge of the physician.

The precise dose and regime to be employed will also depend on the routeof administration, the seriousness of the disease and each patient'scircumstance and age, and should be decided according to the judgment ofthe physician.

When the anti-viral compound comprises ganciclovir, in specificembodiments, the ganciclovir can be administered as its sodium salt. Ina specific embodiment, the ganciclovir is administered to the humanpatient intravenously (e.g., by constant-rate intravenous infusion) orintravitreally (e.g., by intravitreal injection). In a specificembodiment, the intravenous administration of ganciclovir (e.g., byconstant-rate intravenous infusion) comprises administering ganciclovirat about 5 mg per kg per dose every 12 hours. In another specificembodiment, the intravenous administration of ganciclovir (e.g., byconstant-rate intravenous infusion) comprises administering ganciclovirat about 5 mg per kg per dose per day. In another specific embodiment,the intravenous administration of ganciclovir (e.g., by constant-rateintravenous infusion) comprises administering ganciclovir at about 6 mgper kg per dose per day. In another specific embodiment, the intravenousadministration of ganciclovir (e.g., by constant-rate intravenousinfusion) comprises administering ganciclovir at about 2.5 mg per kg perdose every 12 hours. In another specific embodiment, the intravenousadministration of ganciclovir (e.g., by constant-rate intravenousinfusion) comprises administering ganciclovir at about 2.5 mg per kg perdose per day. In another specific embodiment, the intravenousadministration of ganciclovir (e.g., by constant-rate intravenousinfusion) comprises administering ganciclovir at about 1.25 mg per kgper dose per day. In another specific embodiment, the intravenousadministration of ganciclovir (e.g., by constant-rate intravenousinfusion) comprises administering ganciclovir at about 1.25 mg per kgper dose and 3 doses per week. In a specific embodiment, the intravenousadministration of ganciclovir (e.g., by constant-rate intravenousinfusion) comprises administering ganciclovir at about 5 mg per kg perdose every 12 hours for about 14 to 21 days, and then at about 5 mg perkg per dose per day and 7 days per week. In another specific embodiment,the intravenous administration of ganciclovir (e.g., by constant-rateintravenous infusion) comprises administering ganciclovir at about 5 mgper kg per dose every 12 hours for about 14 to 21 days, and then atabout 6 mg per kg per dose per day and 5 days per week. In a specificembodiment, the intravitreal administration of ganciclovir (e.g., byintravitreal injection) comprises administering ganciclovir at about 2mg to 5 mg per dose (e.g., in a volume of 0.1 mL). In another specificembodiment, the intravitreal administration of ganciclovir (e.g., byintravitreal injection) comprises administering ganciclovir at about 2mg per dose (e.g., in a volume of 0.1 mL). In another specificembodiment, the intravitreal administration of ganciclovir (e.g., byintravitreal injection) comprises administering ganciclovir at about 3mg per dose (e.g., in a volume of 0.1 mL). In another specificembodiment, the intravitreal administration of ganciclovir (e.g., byintravitreal injection) comprises administering ganciclovir at about 4mg per dose (e.g., in a volume of 0.1 mL). In another specificembodiment, the intravitreal administration of ganciclovir (e.g., byintravitreal injection) comprises administering ganciclovir at about 5mg per dose (e.g., in a volume of 0.1 mL). The frequency of intravitrealadministration (e.g., by intravitreal injection) of ganciclovir can bedetermined by the location, severity, and response of the disease, andtolerance of the human patient to the ganciclovir treatment, and canrange from, for example, every other day to biweekly. In a specificembodiment, the intravitreal administration of ganciclovir (e.g., byintravitreal injection) comprises administering ganciclovir biweekly. Inanother specific embodiment, the intravitreal administration ofganciclovir (e.g., by intravitreal injection) comprises administeringganciclovir weekly. In another specific embodiment, the intravitrealadministration of ganciclovir (e.g., by intravitreal injection)comprises administering ganciclovir every other day. When the humanpatient has an initial presentation of CMV retinitis and/or is in avision threatening situation (e.g., optic nerve and/or maculathreatening), in a specific embodiment, the intravitreal administrationof ganciclovir (e.g., by intravitreal injection) comprises administeringganciclovir biweekly. Intravitreal administration (e.g., by intravitrealinjection) of ganciclovir may or may not be performed in conjunctionwith systemic therapy with ganciclovir or other anti-viral compounds.Intravitreal administration (e.g., by intravitreal injection) ofganciclovir may or may not be performed in conjunction with intravitrealadministration (e.g., by intravitreal injection) of other anti-viralcompounds. The precise dose of ganciclovir and regime to be employed canalso be adjusted depending on the route of administration, theseriousness of the disease and the patient's circumstance and age, andcan be decided according to the judgment of the physician.

When the anti-viral compound comprises valganciclovir, in specificembodiments, the valganciclovir can be administered as valganciclovirhydrochloride. In a specific embodiment, the valganciclovir isadministered to the human patient orally (e.g., in the form of a tabletor oral solution). In a specific embodiment, the oral administration ofvalganciclovir (e.g., in the form of a tablet or oral solution)comprises administering valganciclovir at about 900 mg per dose and twodoses per day. In another specific embodiment, the oral administrationof valganciclovir (e.g., in the form of a tablet or oral solution)comprises administering valganciclovir at about 900 mg per dose per day.In another specific embodiment, the oral administration ofvalganciclovir (e.g., in the form of a tablet or oral solution)comprises administering valganciclovir at about 450 mg per dose and twodoses per day. In another specific embodiment, the oral administrationof valganciclovir (e.g., in the form of a tablet or oral solution)comprises administering valganciclovir at about 450 mg per dose per day.In another specific embodiment, the oral administration ofvalganciclovir (e.g., in the form of a tablet or oral solution)comprises administering valganciclovir at about 450 mg per dose and onedose every two days. In another specific embodiment, the oraladministration of valganciclovir (e.g., in the form of a tablet or oralsolution) comprises administering valganciclovir at about 450 mg perdose and two doses every week. In a specific embodiment, the oraladministration of valganciclovir (e.g., in the form of a tablet or oralsolution) comprises administering valganciclovir at about 900 mg perdose and two doses per day for 21 days, and then at about 900 mg perdose per day. When the human patient is an adult patient who hasreceived a heart or kidney-pancreas transplant, in a specificembodiment, the oral administration of valganciclovir (e.g., in the formof a tablet or oral solution) comprises administering valganciclovir atabout 900 mg per dose per day starting within 10 days of transplantationuntil about 100 days post-transplantation. When the human patient is anadult patient who has received a kidney transplant, in a specificembodiment, the oral administration of valganciclovir (e.g., in the formof a tablet or oral solution) comprises administering valganciclovir atabout 900 mg per dose per day starting within 10 days of transplantationuntil about 200 days post-transplantation. The precise dose ofvalganciclovir and regime to be employed can also be adjusted dependingon the route of administration, the seriousness of the disease and thepatient's circumstance and age, and can be decided according to thejudgment of the physician.

When the anti-viral compound comprises foscarnet, in specificembodiments, the foscarnet can be administered as its sodium salt. In aspecific embodiment, the foscarnet is administered to the human patientintravenously (e.g., by controlled intravenous infusion) orintravitreally (e.g., by intravitreal injection). In a specificembodiment, the intravenous administration of foscarnet (e.g., bycontrolled intravenous infusion) comprises administering foscarnet atabout 90 mg per kg per dose every 12 hours. In another specificembodiment, the intravenous administration of foscarnet (e.g., bycontrolled intravenous infusion) comprises administering foscarnet atabout 60 mg per kg per dose every 8 hours. In another specificembodiment, the intravenous administration of foscarnet (e.g., bycontrolled intravenous infusion) comprises administering foscarnet atabout 90 mg per kg per dose per day. In another specific embodiment, theintravenous administration of foscarnet (e.g., by controlled intravenousinfusion) comprises administering foscarnet at about 120 mg per kg perdose per day. In a specific embodiment, the intravenous administrationof foscarnet (e.g., by controlled intravenous infusion) comprisesadministering foscarnet at about 90 mg per kg per dose every 12 hours orat about 60 mg per kg per dose every 8 hours for about 2 to 3 weeks, andthen at about 90 mg per kg per dose per day. In another specificembodiment, the intravenous administration of foscarnet (e.g., bycontrolled intravenous infusion) comprises administering foscarnet atabout 90 mg per kg per dose every 12 hours or at about 60 mg per kg perdose every 8 hours for about 2 to 3 weeks, and then at about 120 mg perkg per dose per day. In a specific embodiment, the intravitrealadministration of foscarnet (e.g., by intravitreal injection) comprisesadministering foscarnet at about 2.4 mg per dose (e.g., in a volume of0.1 mL). The frequency of intravitreal administration (e.g., byintravitreal injection) of foscarnet can be determined by the location,severity, and response of the disease, and tolerance of the humanpatient to the foscarnet treatment, and can range from, for example,every other day to biweekly. In a specific embodiment, the intravitrealadministration of foscarnet (e.g., by intravitreal injection) comprisesadministering foscarnet biweekly. In another specific embodiment, theintravitreal administration of foscarnet (e.g., by intravitrealinjection) comprises administering foscarnet weekly. In another specificembodiment, the intravitreal administration of foscarnet (e.g., byintravitreal injection) comprises administering foscarnet every otherday. When the human patient has an initial presentation of CMV retinitisand/or is in a vision threatening situation (e.g., optic nerve and/ormacula threatening), in a specific embodiment, the intravitrealadministration of foscarnet (e.g., by intravitreal injection) comprisesadministering foscarnet biweekly. Intravitreal administration (e.g., byintravitreal injection) of foscarnet may or may not be performed inconjunction with systemic therapy with foscarnet or other anti-viralcompounds. Intravitreal administration (e.g., by intravitreal injection)of foscarnet may or may not be performed in conjunction withintravitreal administration (e.g., by intravitreal injection) of otheranti-viral compounds. The precise dose of foscarnet and regime to beemployed can also be adjusted depending on the route of administration,the seriousness of the disease and the patient's circumstance and age,and can be decided according to the judgment of the physician.

When the anti-viral compound comprises cidofovir, in specificembodiments, the cidofovir is administered to the human patientintravenously (e.g., by infusion). In a specific embodiment, theintravenous administration of cidofovir (e.g., by infusion) comprisesadministering cidofovir at about 5 mg per kg per dose per week. Inanother specific embodiment, the intravenous administration of cidofovir(e.g., by infusion) comprises administering cidofovir at about 5 mg perkg per dose and one dose every 2 weeks. In a specific embodiment, theintravenous administration of cidofovir (e.g., by infusion) comprisesadministering cidofovir at about 5 mg per kg per dose per week for about2 weeks, and then at about 5 mg per kg per dose and one dose every 2weeks. The precise dose of cidofovir and regime to be employed can alsobe adjusted depending on the route of administration, the seriousness ofthe disease and the patient's circumstance and age, and can be decidedaccording to the judgment of the physician.

When the anti-viral compound comprises leflunomide, in specificembodiments, the leflunomide is administered to the human patient orally(e.g., in the form of a tablet). In a specific embodiment, the oraladministration of leflunomide (e.g., in the form of a tablet) comprisesadministering leflunomide at about 100 mg per dose per day. In anotherspecific embodiment, the oral administration of leflunomide (e.g., inthe form of a tablet) comprises administering leflunomide at about 20 mgper dose per day. In another specific embodiment, the oraladministration of leflunomide (e.g., in the form of a tablet) comprisesadministering leflunomide at about 10 mg per dose per day. In a specificembodiment, the oral administration of leflunomide (e.g., in the form ofa tablet) comprises administering leflunomide at about 100 mg per doseper day for 3 days, and then at about 20 mg per dose per day. In anotherspecific embodiment, the oral administration of leflunomide (e.g., inthe form of a tablet) comprises administering leflunomide at about 100mg per dose per day for 3 days, and then at about 10 mg per dose perday. The precise dose of leflunomide and regime to be employed can alsobe adjusted depending on the route of administration, the seriousness ofthe disease and the patient's circumstance and age, and can be decidedaccording to the judgment of the physician.

In embodiments wherein the human patient is treated with both apopulation of allogeneic T cells comprising CMV-specific T cells and ananti-viral compound as described herein, the population of allogeneic Tcells comprising CMV-specific T cells and the antiviral compound can beadministered to the human patient concurrently, for example, at aboutthe same time, at the same day, or same week, or same multi-week periodduring which the population of allogeneic T cells is administeredweekly, or on similar dosing schedules, or on different but overlappingdosing schedules.

In specific embodiments, the anti-viral compound can be administeredbefore (e.g., about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months,5 months, 6 months, 7 months or 8 months before) or after (e.g., about 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7months or 8 months after) the administration of the population ofallogeneic T cells comprising CMV-specific T cells.

The term “about” shall be construed so as to allow normal variation.

5.6. Patients

The human patient can be anyone who has CMV retinitis (e.g., diagnosedby ophthalmologic examination) and who is infected with HIV or has beenthe recipient of a solid organ transplant. In specific embodiments, thehuman patient has an active, not latent, CMV infection.

In specific embodiments, a CMV in the human patient has at least onemutation in its genome that confers resistance to one or more anti-viralagents. In a specific embodiment, the one or more anti-viral agents areselected from the group consisting of ganciclovir, foscarnet,valganciclovir, cidofovir, leflunomide, and combinations thereof. In aspecific embodiment, the mutation is in the UL97 (cytomegalovirus viralphosphotransferase) gene. In a further specific embodiment, the mutationin the UL97 gene confers resistance to ganciclovir and valganciclovir.In another specific embodiment, the mutation is in the UL54(cytomegalovirus DNA polymerase) gene. In another further specificembodiment, the mutation in the UL54 gene confers resistance tofoscarnet and cidofovir. In another further specific embodiment, themutation in the UL54 gene confers resistance to ganciclovir,valganciclovir, foscarnet and cidofovir. In another specific embodiment,a first mutation is in the UL97 gene and a second mutation is in theUL54 gene. In another further specific embodiment, a first mutation isin the UL97 gene and a second mutation is in the UL54 gene, wherein thefirst mutation in the UL97 gene confers resistance to ganciclovir andvalganciclovir, and the second mutation in the UL54 gene confersresistance to foscarnet and cidofovir. In another further specificembodiment, a first mutation is in the UL97 gene and a second mutationis in the UL54 gene, wherein the first mutation in the UL97 gene confersresistance to ganciclovir and valganciclovir, and the second mutation inthe UL54 gene confers resistance to ganciclovir, valganciclovir,foscarnet and cidofovir.

In certain embodiments, the methods of treating CMV retinitis furthercomprise prior to said administering step a step of genotyping a CMV ofthe human patient. In specific embodiments, the methods of treating CMVretinitis further comprise genotyping a CMV of the human patient toidentify at least one mutation (e.g., mutation(s) in the UL97 geneand/or mutation(s) in the UL54 gene) that confers resistance to one ormore anti-viral agents, before the administering of the population ofallogeneic T cells comprising CMV-specific T cells. CMV can be genotypedby any method known in the art, for example, by PCR or DNA sequencing,and can use any infected cell or tissue sample from the patient (e.g., acell/tissue sample from the eye or blood). The genotyping can beperformed using any commercially available kit for genotyping CMV or inany clinical reference laboratory.

CMV retinitis is an inflammation of the retina caused by humancytomegalovirus, which leads to progressive loss of vision andblindness, and usually occurs in immunocompromised patients.

In specific embodiments, the human patient has completely lost his/hervision. In specific embodiments, the human patient has lost more than90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of his/her vision. Inspecific embodiments, the human patient has lost about 90%, 80%, 70%,60%, 50%, 40%, 30%, 20%, or 10% of his/her vision. In specificembodiments, the human patient has improved vision (for example, byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%,250%, 300%, 400% or 500%, or by more than 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400% or 500%) after beingadministered the population of allogeneic T cells comprisingCMV-specific T cells. In specific embodiments, the human patient hasstabilized vision after being administered the population of allogeneicT cells comprising CMV-specific T cells. In specific embodiments, thehuman patient has complete resolution of retinal inflammation afteradministered the population of allogeneic T cells comprisingCMV-specific T cells. In specific embodiments, the human patient haspartial resolution of retinal inflammation after administered thepopulation of allogeneic T cells comprising CMV-specific T cells.

In alternative embodiments, the human patient is infected with HIV, orhas been the recipient of a solid organ transplant from a transplantdonor.

In certain embodiments, the human patient is infected with HIV. Inspecific embodiments, the human patient has AIDS. In a specificembodiment, the human patient is an AIDS patient with mid to higherlevel viral loads. In specific embodiments, the human patient does notdevelop uveitis after administration of the population of allogeneic Tcells comprising CMV-specific T cells.

In certain embodiments, the human patient has been the recipient of asolid organ transplant from a transplant donor. In specific embodiments,the solid organ transplant that the human patient has received is akidney transplant, a liver transplant, a heart transplant, an intestinaltransplant, a pancreas transplant, a lung transplant, a small boweltransplant, or a combination thereof. In a specific embodiment, thesolid organ transplant that the human patient has received is a kidneytransplant. In specific embodiments wherein the human patient has beenthe recipient of a solid organ transplant from a transplant donor, thepopulation of allogeneic T cells is derived from a donor other than thetransplant donor. In specific embodiments, the human patient does notdevelop uveitis after administration of the population of allogeneic Tcells comprising CMV-specific T cells. In specific embodiments, thehuman patient does not develop organ allograft rejection afteradministration of the population of allogeneic T cells comprisingCMV-specific T cells.

In specific embodiments, the human patient has not been the recipient ofa hematopoietic stem cell transplant (e.g., a bone marrow transplant, aperipheral blood stem cell transplant, or a cord blood transplant).

In specific embodiments, the human patient has failed a previous therapyto treat the CMV retinitis. A human patient is considered to have faileda therapy to treat the CMV retinitis if the CMV retinitis is resistantto the therapy and/or if the human patient has been taken off thetherapy due to intolerance of the therapy (for example, due to toxicityof the therapy in view of the patient's age or condition). CMV retinitisis considered resistant to a therapy, if the CMV retinitis has noresponse, or has an incomplete response (a response that is less than acomplete remission), or progresses, or relapses after the therapy. Acomplete remission is a complete resolution of all clinical evidence ofthe disease, optionally confirmed by ophthalmologic examination,lasting, for example, for at least three weeks following completion ofthe therapy. In specific embodiments, the previous therapy is treatmentwith at least one anti-viral agent. In a specific embodiment, the atleast one anti-viral agent is selected from the group consisting ofganciclovir, foscarnet, valganciclovir, cidofovir, leflunomide, andcombinations thereof. In a specific embodiment, the at least oneanti-viral agent has been administered systematically. In a specificembodiment, the at least one anti-viral agent has been administeredlocally to the eye. In specific embodiments, the previous therapy is atherapy to recover or increase the immune function of the human patient.

EXAMPLE

Certain embodiments provided herein are illustrated by the followingnon-limiting example, which demonstrates that the therapy with apopulation of allogeneic T cells comprising CMV-specific T cellsaccording to the invention is effective in treating CMV retinitis in ahuman patient who is infected with HIV or who has been the recipient ofa solid organ transplant.

6.1. Introduction

Although it is reassuring that intravenously infused donor derived Tcells can cross the blood brain barrier and enter the CNS, it could notbe assumed from prior studies that the T-cells would cross theblood-retina barrier for effective treatment of CMV retinitis. Assumingthat T-cells would enter the retina, a further concern for treatment ofretinitis with adoptively transferred T-cells would be the potential forinducing immune recovery uveitis, which is an entity well described inAIDS patients with CMV retinitis treated with HAART, who develop uveitisupon recovery of immune function (Jabs et al., 2015, Ophthalmologypii:S0161-6420(15)00175-X, published online Apr. 16, 2015). The use ofthird party CMV-specific T-cells for the treatment of retinitis raisesadditional concerns about appropriate homing of the infused T-cells tothe eye, and of additional potential risks with respect to inducinguveitis.

The development of retinitis and corresponding immune control ofinfection have thus far not been perfectly correlated. For example,paradoxically, active HCMV retinitis has been reported in the presenceof persistently high CD4+ T-cell counts during HAART. Although thismight be explained by the low number of circulating HCMV-specific CD8+ Tcells, it has not been clearly shown (Lin et al., 2002, Retina22:268-277). The study described herein and recent observations byothers provide evidence that CMV viral load during retinitis does notnecessarily correlate with disease progression, which is in contrast toother HCMV diseases, such as pneumonitis or colitis. Furthermore,several reports in HIV patients have shown that, in some cases, therestoration of immune activity by antiretroviral (HAART) therapy mightbe associated with the development of retinitis. In such cases, thepresumption would be that the retina was latently infected with CMVthroughout, but became inflamed with the infiltration of immune cellsafter HAART.

6.2. Methods:

All cellular products were processed in the GMP facility at MemorialSloan Kettering Cancer Center under standard SOPs and FDA compliantprotocols.

The generation and characterization of CMVpp65 epitope specific T-cellshas been detailed in the recently published manuscript (Koehne et al.,2015, Biol Blood Marrow Transplant pii: S1083-8791(15)00372-9, publishedonline May 29, 2015). CMVpp65 specific T-cells were generated fromCMV-seropositive healthy marrow transplant donors by sensitization invitro with autologous, cytokine-activated monocytes (CAMS) loaded with apool of synthetic 15-mer peptides spanning the sequence of CMV proteinpp65. Autologous transplant donor-derived CAMS and Epstein-Barr virustransformed B lymphocyte cell lines (EBV-BLCLs) were generated aspreviously described (Trivedi et al., 2005, Blood 105:2793-2801; Hasanet al., 2009, J Immunol 183:2837-2850).

CMV-specific T cells were infused as per the clinical protocol accordingto the treatment schema outlined in FIG. 1. T cells were chosen from aGMP grade T cell bank from donors who had consented to the third partyuse of their T-cells. T-cells matching the patient for at least 2 HLAalleles that demonstrated anti-CMV cytotoxic activity restricted by anHLA allele shared with the patient were selected for infusion. Initialtreatment consisted of 3 weekly infusions of 1×10⁶ T-cells/kg. This wasfollowed by an observation period of 3 weeks, after which patients couldreceive another cycle of 3 weekly infusions based on response. As shownin FIG. 2, patients received 1-3 cycles of CMV-specific T-cells, thatwere appropriately matched for 2 or more HLA alleles and appropriatelyrestricted for CMV activity. Blood samples from patients were monitoredat specific intervals post infusion for immune function as well asdetection of CMV-specific T-cells.

6.3. Results:

Immunosuppressed patients developing progressive retinitis despitetreatment with antiviral drugs are in imminent danger of losing visionvery quickly. Banked third party donor derived CMV-specific T-cellsconstitutes an immediately available therapy for such patients. Sixpatients with clinically documented CMV retinitis were treated usingadoptively transferred third party donor derived CMV-specific T-cellscomprising cytotoxic T cells (CTLs) under an IRB (Institutional ReviewBoard) approved protocol. Four of these patients were BMT recipients,one was a kidney transplant recipient, and one was an HIV-positivepatient.

Patient #5 (see clinical chronology of FIG. 3) is a 68-year-old male whoreceived a kidney transplant, and developed CMV retinitis. He had ahistory of Wegener's granulomatosis nephropathy status post kidneytransplant, and was on chronic immunosuppressive therapy (prednisone,tacrolimus, and mycophenolate) for graft rejection prophylaxis. Hisophthalmologic course was also complicated by glaucoma requiring tubeshunt OD (oculus dexter, i.e., right eye), tube shunt revision/CE/IOL(cataract extraction with intraocular lens implant) OD, tubeshunt/CE/IOL OS (oculus sinister, i.e., left eye), 2nd tube shuntsurgery OS, diode laser cyclophotocoagulation OD. The patient could nottolerate aggressive systemic antiviral therapy due to kidney toxicity ofthose agents (foscarnet and ganciclovir), and therefore was treated withintravitreal injections of ganciclovir and foscarnet, and leflunamide,but the patient did not respond. The patient was also treated withvalganciclovir. In addition, he developed peripheral neuropathy due to aside effect of leflunomide. It was found that the CMV of this patienthad a mutation in UL97, conferring resistance to ganciclovir andvalganciclovir. He was therefore referred for CMV specific T-celltherapy. The patient received two cycles of CMV specific T cells for atotal of 6 doses of T cells. Fluid was aspirated from his eye duringtreatment, and demonstrated complete clearance of virus from the eye(FIG. 4, Table 1).

Patient #6 is a 56-year-old male with HIV infection and CMV retinitis.The patient progressed after treatment with systemic antivirals followedby biweekly, and then weekly injections of antivirals in the eye for 2-3months. The patient received injections of antivirals in the eye twice aweek, and became intolerant of these frequent injections. He wastherefore referred for CMV specific T-cell therapy. This patientreceived one cycle of CMV specific T cells for a total of 3 doses of Tcells and had a complete clearance of retinal disease (Table 1).

TABLE 1 Patient #5 and patient #6. # Rounds Age Underlying MedicalT-cell VA F/u Recurrent Retinal Patient (yr) Diagnosis InfusionsPre-T-cells Final VA (mth) Retinitis Uveitic Episodes detachment #5 68Renal transplant on 2 CF OD 3/200 OD 8 None None None immunosuppressantsfor graft rejection prophylaxis #6 56 HIV with chronically 1 20/25 OD20/20 14 None Cystoid macular None suppressed CD4 edema OD 7 count <50despite months after HAART; history of infusions (no lymphoma s/p chemoretinitis, CMV PCR negative, self-resolved) VA: visual acuity. CF: countfingers (worse than 20/400). F/u: follow up. mth: month. s/p: statuspost. OD: oculus dexter, i.e., right eye.

Four of the 6 treated patients, including the one HIV-positive patientand the one kidney transplant recipient, had a complete response totreatment with complete resolution of retinal inflammation onophthalmologic examination. The vision improved in these patients, orstabilized. One BMT patient had a partial response to treatment. Hereceived 2 cycles of treatment after which the response was sustainedwith no deterioration. One BMT patient received only one dose ofCMV-specific T cells and could not continue, and is therefore notevaluable.

No GvHD was observed in any of the treated patients. Patient #5 has notdemonstrated any evidence of uveitis. The patient with HIV (#6) had acomplete response to treatment. This patient transiently developedvitreal inflammation which resolved without sequelae and with completeresolution of CMV retinitis. The cystoid macular edema that developed inpatient #6 was apparently due to a worsening of a pre-existingepiretinal membrane (macular pucker), not secondary to the T celltherapy or CMV retinitis activation.

CMV-specific T-cells were enumerated from the patients at different timepoints post infusion. An expansion in the numbers of CMV-specificT-cells could be demonstrated in all responding patients, including thepatient with a partial response. As shown in FIG. 5 for a representativeexample (patient #5), increases in CMV-specific T cells were associatedwith decline in CMV viral load in blood. In the HIV-positive patient,functional CMV-specific T-cells were able to be detected for 6 weekspost infusion of the last dose of T-cells.

INCORPORATION BY REFERENCE

Various publications are cited herein, the disclosures of which arehereby incorporated by reference herein in their entireties.

1. A method of treating CMV (cytomegalovirus) retinitis in a humanpatient in need thereof, comprising administering to the human patient apopulation of allogeneic T cells comprising CMV-specific T cells;wherein the human patient is infected with HIV, and wherein thepopulation of allogeneic T cells is restricted by an HLA allele sharedwith at least some, optionally all, CMV-infected cells in the patient.2. (canceled)
 3. A method of treating CMV retinitis in a human patientin need thereof, comprising administering to the human patient apopulation of allogeneic T cells comprising CMV-specific T cells;wherein the human patient has been the recipient of a solid organtransplant from a transplant donor, and wherein the population ofallogeneic T cells is restricted by an HLA allele shared with at leastsome, optionally all, CMV-infected cells in the patient.
 4. The methodof claim 3, wherein the solid organ transplant is a kidney transplant, aliver transplant, a heart transplant, an intestinal transplant, apancreas transplant, a lung transplant, a small bowel transplant, or acombination thereof. 5-12. (canceled)
 13. The method of claim 1, whichfurther comprises prior to said administering step a step of generatingthe population of allogeneic T cells in vitro.
 14. The method of claim13, wherein the step of generating the population of allogeneic T cellsin vitro comprises sensitizing allogeneic T cells to one or more CMVantigens. 15-29. (canceled)
 30. The method of claim 1, wherein theadministering is by infusion of the population of allogeneic T cells.31-40. (canceled)
 41. The method of claim 1, further comprising, afteradministering to the human patient the population of allogeneic T cells,administering to the human patient a second population of allogeneic Tcells comprising CMV-specific T cells; wherein the second population ofallogeneic T cells is restricted by a different HLA allele shared withat least some, optionally all, of the CMV-infected cells. 42-44.(canceled)
 45. The method of claim 1, wherein the human patient hasfailed a previous therapy to treat the CMV retinitis. 46-47. (canceled)48. The method of claim 45, wherein the previous therapy is treatmentwith at least one anti-viral agent.
 49. The method of claim 48, whereinthe at least one anti-viral agent is selected from the group consistingof ganciclovir, foscarnet, valganciclovir, cidofovir, leflunomide, andcombinations thereof. 50-51. (canceled)
 52. The method of claim 1,wherein the population of allogeneic T cells has not been transduced exvivo with a gene that encodes a CMV-specific T-cell receptor.
 53. Themethod of claim 1, wherein at least some, optionally all, of the cellsof the population of allogeneic T cells are rapamycin-sensitive.
 54. Themethod of claim 1, wherein the population of allogeneic T cells is notadministered in combination with a PD-1 antagonist.
 55. The method ofclaim 1, wherein the human patient has not been the recipient of ahematopoietic stem cell transplant.
 56. The method of claim 1, whereinthe human patient has an active, not latent, CMV infection.
 57. Themethod of claim 1, wherein a CMV in the human patient has at least onemutation in its genome that confers resistance to one or more anti-viralagents.
 58. The method of claim 57, wherein the one or more anti-viralagents are selected from the group consisting of ganciclovir, foscarnet,valganciclovir, cidofovir, leflunomide, and combinations thereof. 59.The method of claim 57, wherein the at least one mutation is a mutationin the UL97 gene, a mutation in the UL54 gene, or a first mutation inthe UL97 gene and a second mutation in the UL54 gene. 60-61. (canceled)62. The method of claim 57, which further comprises prior to saidadministering step a step of genotyping a CMV of the human patient.